Mutation of Potential Phosphorylation Sites in the Recombinant R Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Has Significant Effects on Domain Conformation

1995 ◽  
Vol 206 (1) ◽  
pp. 207-214 ◽  
Author(s):  
A.M. Dulhanty ◽  
X.B. Chang ◽  
J.R. Riordan
Keyword(s):  
Cystic Fibrosis ◽  
Phosphorylation Sites ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

Functional dissection of the R domain of cystic fibrosis transmembrane conductance regulator 1

FEBS Letters ◽  
1999 ◽  
Vol 445 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Jason E. Tasch ◽  
Bryan Zerhusen ◽  
Jiying Zhao ◽  
Jianjie Ma ◽  
Pamela B. Davis
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

scholarly journals Domain-domain associations in cystic fibrosis transmembrane conductance regulator

2002 ◽  
Vol 282 (5) ◽  
pp. C1170-C1180 ◽  
Author(s):  
Wenlan Wang ◽  
Zhaoping He ◽  
Thomas J. O'Shaughnessy ◽  
John Rux ◽  
William W. Reenstra
Keyword(s):  
Cystic Fibrosis ◽  
Fluorescence Quenching ◽  
Tryptophan Fluorescence ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Tryptophan Fluorescence Quenching ◽  
Overlay Assay ◽  
R Domain

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione- S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic dig estion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.

CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain

1997 ◽  
Vol 273 (1) ◽  
pp. L127-L133 ◽  
Author(s):  
D. J. Wilkinson ◽  
T. V. Strong ◽  
M. K. Mansoura ◽  
D. L. Wood ◽  
S. S. Smith ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Phosphorylation Sites ◽  
Transmembrane Conductance Regulator ◽  
Additive Effects ◽  
Transmembrane Conductance ◽  
Phosphorylation State ◽  
Consensus Sequences ◽  
Maximal Activation ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (KA) for activation by 3-isobutyl-1-methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.

scholarly journals Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains

2006 ◽  
Vol 282 (7) ◽  
pp. 4533-4544 ◽  
Author(s):  
Wei Wang ◽  
Karen Bernard ◽  
Ge Li ◽  
Kevin L. Kirk
Keyword(s):  
Cystic Fibrosis ◽  
Nucleotide Binding ◽  
Salt Transport ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Channel Opening ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.

scholarly journals Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain

2004 ◽  
Vol 378 (1) ◽  
pp. 151-159 ◽  
Author(s):  
L. Daniel HOWELL ◽  
Roy BORCHARDT ◽  
Jolanta KOLE ◽  
Andrew M. KAZ ◽  
Christoph RANDAK ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Atp Hydrolysis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A ◽  
R Domain

Gating of the CFTR Cl− channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1–R–GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1–R–GST by increasing the apparent Km for ATP (from 70 to 250 µM) and the Hill coefficient (from 1 to 1.7) without changing the Vmax. When fusion proteins were photolabelled with 8-azido-[α-32P]ATP, PKA phosphorylation increased the apparent kd for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1–R–GST but not of R–GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2–MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1–R–GST. Thus when the R domain in NBD1–R–GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.

Sign in / Sign up

Export Citation Format

Share Document